Abstract

The target of rapamycin (TOR) is a conserved protein kinase and a central controller of growth. TOR can be part of two structurally and functionally distinct complexes, termed TOR complex 1 and TOR complex 2. Mammalian TOR complex 2 (mTORC2) is composed of mTOR, Rictor, Sin1 and mLST8. Both mTORC1 and mTORC2 are activated by growth factors. The mechanism via which growth factors regulate mTORC2 has been elusive until recently. mTORC2 binds ribosomes in a growth factor stimulated manner and this association is required for mTORC2 activity. mTOR complex 2 functions include control of spatial cell growth and metabolism and thus, mTORC2 deregulation has been linked to various disorders including cancer and diabetes. mTORC2 phosphorylates and thereby activates the AGC kinase family member Akt (PKB). Akt has many different targets and functions, not all of which depend on mTORC2 mediated Akt phosphorylation. In order to gain a better understanding of mTORC2 function, we asked where mTORC2 signaling is localized. A number of studies localized mTORC2, functionally or physically, either to the endoplasmic reticulum (ER) or to mitochondria.We investigated whether these seemingly unrelated observations concerning mTORC2 localization, might be the consequence of mTORC2 signaling at MAM. MAM or mitochondria-associated ER membrane is a quasi-synaptic subdomain between the ER and mitochondria. MAM plays a crucial role in the regulation of mitochondrial metabolism and cell survival by gating both the calcium flux and phospholipid trafficking between the ER and mitochondria. First, we analyzed mTORC2 subcellular localization. mTORC2 is localized to the ER adjacent to mitochondria and mTORC2 can be biochemically isolated from MAM structures. mTOR complex 2 interacts with the IP3R-Grp75-VDAC1 complex, a tether that connects ER and mitochondria at MAM. Insulin stimulates mTORC2 localization to MAM and mTORC2 interaction with the IP3R-Grp75-VDAC1 complex. MAM localization of mTORC2 depends on mTORC2-ribosome interaction. Next we investigated the function of mTORC2 at MAM. Rictor (mTORC2) knockout causes a decrease in MAM formation. Growth factors stimulate MAM formation via mTORC2 and the Akt substrate PACS2, a MAM resident protein. As expected for MAM deficient cells, mTORC2 disruption changes the calcium flux from the ER to mitochondria at MAM. Furthermore, we observe a reduction of Akt mediated phosphorylation of the MAM calcium channel IP3R upon Rictor knockout. Thus, mTORC2 signaling at MAM controls MAM mediated calcium release via the Akt targets PACS2 and IP3R. Since MAM disruption and calcium signaling both affect mitochondrial metabolism, we proceeded by analyzing the mitochondrial physiology of mTORC2 deficient cells. Rictor knockout cells exhibit a disruption of VDAC1-HK2 binding, caused by a lack of Akt mediated phosphorylation of HK2 at MAM. This, together with the defect in MAM, induces an increase in basal respiration, mitochondrial inner membrane potential, and ATP production in the mTORC2 deficient cells, culminating in apoptosis. Thus, mTORC2 at MAM appears to control several aspects of mitochondrial physiology. These findings emphasize the role of MAM as a signaling hub that controls cell physiology. By identifying the integral role of mTORC2 at the core of this platform, our results provide new insights on the mechanisms that regulate growth and metabolism. These observations may offer new therapeutic strategies against mTORC2 and MAM driven diseases such as diabetes, Alzheimer’s and cancer.